The invention relates to the cloning of animals.
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Researchers have been developing methods for cloning mammalian animals over the past two decades. These reported methods typically include the steps of (1) isolating a cell, most often an embryonic cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (e.g., the oocyte""s nucleus was previously extracted), and (3) allowing the embryo to mature in vivo.
The first successful nuclear transfer experiment using mammalian cells was reported in 1983, where the pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s). McGrath and Solter, 1983, Science 220:1300-1302. Subsequently, others described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGC). These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells but do not differentiate into a grown animal). Matsui et al., 1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38.
Some publications related to murine pluripotent cells stress the importance of steel factor for converting precursor cells into pluripotent cells. U.S. Pat. Nos. 5,453,357 and 5,670,372, entitled xe2x80x9cPluripotent Embryonic Stem Cells and Methods of Making Same,xe2x80x9d issued to Hogan. These same publications indicate that murine pluripotent cells exhibit strong, uniform alkaline phosphatase staining.
Although murine animals were never clearly cloned from nuclear transfer techniques using embryonic cells, some progress was reported in the field of cloning ovine (sheep) animals. One of the first successful nuclear transfer experiments utilizing ovine embryonic cells as nuclear donors was reported in 1986. Willadsen, 1986, Nature 320:63-65. A decade later, others reported that additional lambs were cloned from ovine embryonic cells. Campbell et al., 1996, Nature 380:64-66 and PCT Publication WO 95/20042. Recently, another lamb was reported to be cloned from ovine somatic mammary tissue. Wilmut et al., 1997, Nature 385:810-813. Some methods for cloning ovine animals focused upon utilizing serum deprived somatic ovine cells and cells isolated from ovine embryonic discs as nuclear donors. PCT Publications WO 96/07732 and WO 97/07669. Other methods for cloning ovine animals involved manipulating the activation state of an in vivo matured oocyte after nuclear transfer. PCT Publication WO 97/07668.
While few lambs were produced, publications that disclose cloned lambs report a cloning efficiency that is, at best, approximately 0.4%. Cloning efficiency, as calculated for the previous estimate, is a ratio equal to the number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs.
Despite the slower progress endemic to the field of cloning bovine animals, a bovine animal was cloned using embryonic cells derived from 2-64 cell embryos. This bovine animal was cloned by utilizing the nuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed by nuclear transfer techniques utilizing the inner cell mass cells of a blastocyst stage embryo. Sims and First, 1993, Theriogenology 39:313 and Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268. In addition, another publication reported that cloned bovine embryos were prepared by nuclear transfer techniques that utilized PGCs isolated from fetal tissue. Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled xe2x80x9cEmbryonic Stem Cell-Like Cells.xe2x80x9d However, the reports demonstrated that cloned PGC-derived bovine embryos never clearly developed past the first trimester during gestation. Similarly, embryonic stem cell (e.g., cell line derived from embryos which are undifferentiated, pluripotent, and can establish a permanent cell line which exhibits a stable karyotype), ESC, derived bovine embryos never developed past fifty-five days, presumably due to incomplete placental development. Stice et al., 1996, Biol. Reprod. 54: 100-110.
Despite the progress of cloning ovine and bovine animals, there remains a great need in the art for methods and materials that increase cloning efficiency. In addition there remains a great need in the art to expand the variety of cells that can be utilized as nuclear donors, especially expanding nuclear donors to non-embryonic cells. Furthermore, there remains a long felt need in the art for karyotypically stable permanent cell lines that can be used for genome manipulation and production of transgenic cloned animals.
The present invention relates to cloning technologies. The invention relates in part to immortalized, totipotent cells useful for cloning animals, the embryos produced from these cells using nuclear transfer techniques, animals that arise from these cells and embryos, and the methods and processes for creating such cells, embryos, and animals.
The present invention provides multiple advantages over the tools and methods currently utilized in the field of mammalian cloning. Such features and advantages include:
(1) Production of cloned animals from virtually any type of cell. The invention provides materials and methods for reprogramming non-totipotent cells into totipotent cells. These non-totipotent cells may be of non-embryonic origin. This feature of the invention allows for the ability to assess the phenotype of an existing animal and then readily establish a permanent cell line for cloning that animal.
(2) Creation of permanent cell lines from virtually any type of cell. Permanent cell lines provide a nearly unlimited source of genetic material for nuclear transfer cloning techniques. In one aspect of the invention, non-totipotent precursor cells can be reprogrammed into totipotent and permanent cells. These non-totipotent precursor cells may be non-embryonic cells. Permanent cell lines provide the advantage of enhancing cloning efficiency due to the lower cellular heterogeneity within the cell lines (e.g., permanent cells that have lower rates of differentiation than primary culture cell lines currently used for cloning). In addition, the permanent cell lines can be manipulated in vitro to produce cells, embryos, and animals whose genomes have been manipulated (e.g., transgenic). Furthermore, permanent cell lines can be more easily stored, transported, and re-established in culture than other types of cell lines.
(3) Enhancement of the efficiency for cloning embryos as a result of utilizing asynchronous, permanent, and karyotypically stable cell lines in a complete in vitro embryo production system.
Cloning efficiency can be expressed by the ratio between the number of embryos resulting from nuclear transfer and the number of nuclear transfers performed to give rise to the embryos. Alternatively, cloning efficiency can be expressed as the ratio between the number of live born animals and the number of nuclear transfers performed to give rise to these animals.
In a first aspect, the invention features a totipotent mammalian cell. Preferably, the totipotent mammalian cell is (1) a cultured cell; (2) a cell cultured in a cell line; and (3) an immortalized cell. In addition, the mammalian cell is preferably an ungulate cell and more preferably a bovine cell.
The term xe2x80x9cmammalianxe2x80x9d or xe2x80x9cmammalxe2x80x9d as used herein refers to any animal of the class Mammalia. A mammalian animal of the invention is preferably an endangered animal, or, more preferably, a farm animal. Most preferably, a mammal is an ungulate.
The term xe2x80x9cnon-ovinexe2x80x9d as used herein refers to any animal other than an animal of the family Ovidae. Members of the family Oviadae include sheep. A non-ovine mammal is any member of the class Mammalia other than an animal of the family Ovidae. Preferable non-ovine animals are ungulate animals and most preferably are bovine and porcine animals.
The term xe2x80x9cungulatexe2x80x9d as used herein refers to a four-legged animal having hooves. In other preferred embodiments, the ungulate is selected from the group consisting of domestic or wild representatives of bovids, ovids, cervids, suids, equids and camelids. Examples of such representatives are cows or bulls, bison, buffalo, sheep, big-hom sheep, horses, ponies, donkeys, mule, deer, elk, carbou, goat, water buffalo, camels, llama, alpaca, and pigs. Especially preferred in the bovine species are Bos taurus, Bos indicus, and Bos buffaloes cows or bulls.
The term xe2x80x9cbovinexe2x80x9d as used herein refers to a family of ruminants belonging to the genus Bos or any closely related genera of the family Bovidae. The family Bovidae includes true antelopes, oxen, sheep, and goats, for example. Preferred bovine animals are the cow and ox. Especially preferred bovine species are Bos taurus, Bos indicus, and Bos buffaloes. Other preferred bovine species are Bos primigenius and Bos longifrons. 
The term xe2x80x9ctotipotentxe2x80x9d as used herein refers to a cell that gives rise to all of the cells in a developing cell mass, such as an embryo, fetus, and animal. In preferres embodiments, the term xe2x80x9ctotipotentxe2x80x9d also refers to a cell that gives rise to all of the cells in an animal. A totipotent cell can give rise to all of the cells of a developing cell mass when it is utilized in a procedure for creating an embryo from one or more nuclear transfer steps. An animal may be an animal that functions ex utero. An animal can exist, for example, as a live born animal. Totipotent cells may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of a head such as by manipulation of a homeotic gene.
The terms xe2x80x9cdeveloping cell massxe2x80x9d as used herein refers to a group of cells in which all cells or a portion of the cells are undergoing cell division. The developing cell mass may be an embryo, a fetus, and/or an animal, for example. The developing cell mass may be dividing in vitro (e.g., in culture) or in vivo (e.g., in utero). The developing cell mass may be a product of one or more nuclear transfer processes or may be the product of oocyte activation (e.g., sperm mediated fertilization).
The term xe2x80x9clive bornxe2x80x9d as used herein preferably refers to an animal that exists ex utero. A xe2x80x9clive bornxe2x80x9d animal may be an animal that is alive for at least one second from the time it exits the maternal host. A xe2x80x9clive bornxe2x80x9d animal may not require the circulatory system of an in utero environment for survival. A xe2x80x9clive bornxe2x80x9d animal may be an ambulatory animal. Such animals can include pre- and post-pubescent animals. In addition, a xe2x80x9clive born animalxe2x80x9d may also be deceased for a certain period of time. As discussed previously, a xe2x80x9clive bornxe2x80x9d animal may lack a portion of what exists in a normal animal of its kind. For example, a xe2x80x9clive bornxe2x80x9d animal may lack a head as a result of the deletion or manipulation of one or more homeotic genes.
The term xe2x80x9ctotipotentxe2x80x9d as used herein is to be distinguished from the term xe2x80x9cpluripotent.xe2x80x9d The latter term refers to a cell that differentiates into a sub-population of cells within a developing cell mass, but is a cell that may not give rise to all of the cells in that developing cell mass. Thus, the term xe2x80x9cpluripotentxe2x80x9d can refer to a cell that cannot give rise to all of the cells in a live born animal.
The term xe2x80x9ctotipotentxe2x80x9d as used herein is also to be distinguished from the term xe2x80x9cchimerxe2x80x9d or xe2x80x9cchimera.xe2x80x9d The latter term refers to a developing cell mass that comprises a sub-group of cells harboring nuclear DNA with a significantly different nucleotide base sequence than the nuclear DNA of other cells in that cell mass. The developing cell mass can, for example, exist as an embryo, fetus, and/or animal.
The term xe2x80x9cimmortalizedxe2x80x9d or xe2x80x9cpermanentxe2x80x9d as used herein in reference to cells refers to cells that have exceeded the Hayflick limit. The Hayflick limit can be defined as the number of cell divisions that occur before a cell line becomes senescent. Hayflick set this limit to approximately 60 divisions for most non-immortalized cells. See, e.g., Hayflick and Moorhead, 1961, Exp. Cell. Res. 25: 585-621; and Hayflick, 1965, Exp. Cell Research 37: 614-636, incorporated herein by reference in their entireties including all figures, tables, and drawings. Therefore, an immortalized cell line can be distinguished from non-immortalized cell lines if the cells in the cell line are able to undergo more than 60 divisions. If the cells of a cell line are able to undergo more than 60 cell divisions, the cell line is an immortalized or permanent cell line. The immortalized cells of the invention. are preferably able to undergo more than 70 divisions, are more preferably able to undergo more than 80 divisions, and are most preferably able to undergo more than 90 cell divisions.
Typically, immortalized or permanent cells can be distinguished from non-immortalized and non-permanent cells on the basis that immortalized and permanent cells can be passaged at densities lower than those of non-immortalized cells. Specifically, immortalized cells can be grown to confluence (e.g., when a cell monolayer spreads across an entire plate) when plating conditions do not allow physical contact between the cells. Hence, immortalized cells can be distinguished from non-immortalized cells when cells are plated at cell densities where the cells do not physically contact one another.
The term xe2x80x9cplatedxe2x80x9d or xe2x80x9cplatingxe2x80x9d as used herein in reference to cells refers to establishing cell cultures in vitro. For example, cells can be diluted in cell culture media and then added to a cell culture plate or cell culture dish. Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities.
The meaning of the term xe2x80x9ccell platingxe2x80x9d can also extend to the term xe2x80x9ccell passaging.xe2x80x9d Immortalized cells of the invention can be passaged using cell culture techniques well known to those skilled in the art. The term xe2x80x9ccell passagingxe2x80x9d can refer to such techniques which typically involve the steps of (1) releasing cells from a solid support and disassociation of these cells, and (2) diluting the cells in fresh media suitable for cell proliferation. Immortalized cells can be successfully grown by plating the cells in conditions where they lack cell to cell contact. Cell passaging may also refer to removing a portion of liquid medium bathing cultured cells and adding liquid medium from another source to the cell culture.
The term xe2x80x9cproliferationxe2x80x9d as used herein in reference to immortalized or permanent cells refers to a group of cells that can increase in size and/or can increase in numbers over a period of time.
The term xe2x80x9cconfluencexe2x80x9d as used herein refers to a group of cells where a large percentage of the cells are physically contacted with at least one other cell in that group. Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided. For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel. The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70% of the total surface area, and most preferably represents about 90% of the total surface area.
The term xe2x80x9cculturexe2x80x9d as used herein in reference to cells refers to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment can be any medium known in the art that is. suitable for maintaining cells in vitro, such as suitable liquid media or agar. Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings. Preferred media are AminoMax(trademark)-C100 Basal Medium (Gibco 1701-082), AminoMax(trademark) C-100 Supplement Medium (Gibco 17002-080), and Knockout(trademark) D-MEM Medium (Gibco 10829-108).
Nearly any type of cell can be placed in cell culture conditions. Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells. Cells may be cultured in suspension and/or in monolayers with a heterogeneous population cells. The term xe2x80x9cheterogeneousxe2x80x9d as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example. Cells may be cultured in suspension and/or in monolayers with feeder cells. The term xe2x80x9cfeeder cellsxe2x80x9d is defined hereafter. In preferred embodiments, cells may be successfully cultured by plating the cells in conditions where they lack cell to cell contact. Cell cultures can also be utilized to establish a cell line.
In preferred embodiments, (1) cultured cells undergo cell division; (2) cells are cultured for greater than 5 hours; (3) cells are cultured for greater than 7 hours; (4) cells are cultured for greater than 10 hours; (5) cells are cultured for greater than 12 hours; (6) cells are cultured for greater than 24 hours; (7) cells are cultured for and greater than 48 hours; (8) cells are cultured greater than 3 days; (9) cells are cultured for greater than 5 days; (10) cells are cultured for greater than 10 days; and (11) cells are cultured for greater than 30 days.
The term xe2x80x9csuspensionxe2x80x9d as used herein refers to cell culture conditions in which the cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art.
The term xe2x80x9cmonolayerxe2x80x9d as used herein refers to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of the cells proliferating in the monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support. Preferably less than 15% of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5% of these cells are not attached to the solid support. Cells can also grow in culture in multilayers. The term xe2x80x9cmultilayerxe2x80x9d as used herein refers to cells proliferating in suitable culture conditions where at least 15% of the cells are indirectly attached to the solid support through an attachment to other cells. Preferably, at least 25% of the cells are indirectly attached to the solid support, more preferably at least 50% of the cells are indirectly attached to the solid support, and most preferably at least 75% of the cells are indirectly attached to the solid support.
The term xe2x80x9csubstantially similarxe2x80x9d as used herein in reference to immortalized bovine cells refers to cells from the same organism and the same tissue. In preferred embodiments, substantially similar also refers to cell populations that have not significantly differentiated. For example, preferably less than 15% of the cells in a population of cells have differentiated, more preferably less than 10% of the cell population have differentiated, and most preferably less than 5% of the cell population have differentiated.
The term xe2x80x9ccell linexe2x80x9d as used herein refers to cultured cells that can be passaged more than once. The invention relates to cell lines that can be passaged more than 2, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 80, 100, and 200 times, or preferably more than any integer between 2 and 200, each number not having been explicitly set forth in the interest of conciseness. The concept of cell passaging is defined previously.
In preferred embodiments, (1) the totipotent cells are not alkaline phosphatase positive; (2) the totipotent cells arise from at least one precursor cell; (3) the precursor cell is isolated from and/or arises from any region of an animal; (4) the precursor cell is isolated from and/or arises from any cell in culture; (5) the precursor cell is selected from the group consisting of a non-embryonic cell, a non-fetal cell, a differentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, an amniotic cell, a fetal fibroblast cell, an ovarian follicular cell, a cumulus cell, an hepatic cell, an endocrine cell, an endothelial cell, an epidermal cell, an epithelial cell, a fibroblast cell, a hematopoietic cell, a keratinocyte, a renal cell, a lymphocyte, a melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an osetoblast, a mesenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the Go stage of the cell cycle; and (6) the precursor cell is preferably isolated and/or arises from a mammalian animal, more preferably an ungulate animal, and most preferably a bovine animal.
The term xe2x80x9calkaline phosphatase positivexe2x80x9d as used herein refers to a detectable presence of cellular alkaline phosphatase. Cells that are not alkaline phosphatase positive do not stain appreciably using a procedure for visualizing cellular alkaline phosphatase. Procedures for detecting the presence of cellular alkaline phosphatase. are well-known to a person of ordinary skill in the art. See, e.g., Matsui et al., 1991, xe2x80x9cEffect of Steel Factor and Leukemia Inhibitory Factor on Murine Primordial Germ Cells in Culture,xe2x80x9d Nature 353: 750-752. Examples of cells that stain appreciably for alkaline phosphatase can be found in the art. See, e.g., U.S. Pat. No. 5,453,357, Entitled xe2x80x9cPluripotent Embryonic Stem Cells and Methods of Making Same,xe2x80x9d issued to Hogan on Sep. 26, 1995, which is incorporated by reference herein in its entirety, including all figures, tables, and drawings.
The term xe2x80x9cprecursor cellxe2x80x9d or xe2x80x9cprecursor cellsxe2x80x9d as used herein refers to a cell or cells used to create a cell line of totipotent cells. The cell line is preferably permanent. Precursor cells can be isolated from any mammal, preferably from an ungulate and more preferably from a bovine animal. The precursor cell or cells may be isolated from nearly any cellular entity. For example, a precursor cell or cells may be isolated from blastocysts, embryos, fetuses, and cell lines (e.g., cell lines established from embryonic cells), preferably isolated from fetuses and/or cell lines established from fetal cells, and more preferably isolated from ex utero animals and/or cell cultures and/or cell lines established from such ex utero animals. An ex utero animal may exist as a newborn animal, adolescent animal, yearling animal, and adult animal. The ex utero animals may be alive or post mortem. The precursor cell or cells may be immortalized or non-immortalized. These examples are not meant to be limiting and a further description of these exemplary precursor cells is provided hereafter.
The term xe2x80x9carises fromxe2x80x9d as used herein refers to the conversion of one or more cells into one or more other cells. For example, a non-totipotent precursor cell can be converted into a totipotent cell by utilizing features of the invention described hereafter. This conversion process can be referred to as a reprogramming step. In another example, a precursor cell can give rise to a feeder layer of cells, as defined hereafter. In addition, the term xe2x80x9carises fromxe2x80x9d can refer to the creation of totipotent embryos from immortalized, totipotent cells of the invention, as described hereafter.
The term xe2x80x9creprogrammingxe2x80x9d or xe2x80x9creprogrammedxe2x80x9d as used herein refers to materials and methods that can convert a non-totipotent cell into an totipotent cell. Distinguishing features between totipotent and non-totipotent cells are described previously. An example of materials and methods for converting non-totipotent cells into totipotent cells is to incubate precursor cells with a receptor ligand cocktail. Receptor ligand cocktails are described hereafter.
The term xe2x80x9cisolatedxe2x80x9d as used herein refers to a cell that is mechanically separated from another group of cells. Examples of a group of cells are a developing cell mass, a cell culture, a cell line, and an animal. These examples are not meant to be limiting and the invention relates to any group of cells.
The term xe2x80x9cnon-embryonic cellxe2x80x9d as used herein refers to a cell that is not isolated from an embryo. Non-embryonic cells can be differentiated or non-differentiated. Non-embryonic cells can refer to nearly any somatic cell, such as cells isolated from an ex utero animal. These examples are not meant to be limiting.
For the purposes of the present invention, the term xe2x80x9cembryoxe2x80x9d or xe2x80x9cembryonicxe2x80x9d as used herein refers to a developing cell mass that has not implanted into the uterine membrane of a maternal host. Hence, the term xe2x80x9cembryoxe2x80x9d as used herein can refer to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, and/or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. Hence, an xe2x80x9cembryonic cellxe2x80x9d is isolated from and/or has arisen from an embryo.
An embryo can represent multiple stages of cell development. For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.
The term xe2x80x9cfetusxe2x80x9d as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus can include such defining features as a genital ridge, for example. A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species. The term xe2x80x9cfetal cellxe2x80x9d as used herein can refer to any cell isolated from and/or has arisen from a fetus or derived from a fetus. The term xe2x80x9cnon-fetal cellxe2x80x9d is a cell that is not derived or isolated from a fetus.
The term xe2x80x9cprimordial germ cellxe2x80x9d as used herein refers to a diploid somatic cell capable of becoming a germ cell. Primordial germ cells can be isolated from the genital ridge of a developing cell mass. The genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art. See, e.g., Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod. Dev. 37: 413-424.
The terms xe2x80x9cembryonic germ cellxe2x80x9d and xe2x80x9cEG cellxe2x80x9d as used herein refers to a cultured cell that has a distinct flattened morphology and can grow within monolayers in culture. An EG cell may be distinct from a fibroblast cell. This EG cell morphology is to be contrasted with cells that have a spherical morphology and form multicellular clumps on feeder layers. Embryonic germ cells may not require the presence of feeder layers or presence of growth factors in cell culture conditions. Embryonic germ cells may also grow with decreased doubling rates when these cells approach confluence on culture plates. Embryonic germ cells of the invention may be totipotent. Embryonic germ cells of the invention may not appreciably stain for alkaline phosphatase. Preferably, embryonic germ cells are established in culture media that contains a significant concentration of glucose.
Embryonic germ cells may be established from a cell culture of nearly any type of precursor cell. Examples of precursor cells are discussed herein, and a preferred precursor cell for establishing an embryonic germ cell culture is a genital ridge cell from a fetus. Genital ridge cells are preferably isolated from porcine fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus. An age of a fetus can be determined as described above. The term xe2x80x9caboutxe2x80x9d with respect to fetuses can refer to plus or minus five days. As described herein, EG cells maybe physically isolated from a primary culture of cells, and these isolated EG cells may be utilized to establish a cell culture that eventually forms a homogenous or nearly homogenous line of EG cells.
The term xe2x80x9cembryonic stem cellxe2x80x9d as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture. Embryonic stem cells may be cultured with or without feeder cells. Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and pre-blastocyst stage embryos. Embryonic stem cells are well known to a person of ordinary skill in the art. See, e.g., WO 97/37009, entitled xe2x80x9cCultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,xe2x80x9d Stice and Golueke, published Oct. 9, 1997, and Yang and Anderson, 1992, Theriogenology 38: 315-335, both of which are incorporated herein by reference in their entireties, including all figures, tables, and drawings.
The term xe2x80x9camniotic cellxe2x80x9d as used herein refers to any cultured or non-cultured cell isolated from amniotic fluid. Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996, Theriogenology 45: 225; Garcia and Salaheddine, 1997, Theriogenology 47:1003-1008; Leibo and Rail, 1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet. Rec. 127: 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings. Particularly preferred are cultured amniotic cells that are spherical (e.g., cultured amniotic cells that do not display a fibroblast-like morphology). Also preferred amniotic cells are fetal fibroblast cells. The terms xe2x80x9cfibroblast,xe2x80x9d fibroblast-like,xe2x80x9d xe2x80x9cfetal,xe2x80x9d and xe2x80x9cfetal fibroblastxe2x80x9d are defined hereafter.
The terms xe2x80x9cfibroblast-likexe2x80x9d and xe2x80x9cfibroblastxe2x80x9d as used herein refer to cultured cells that have a distinct flattened morphology and that are able to grow within monolayers in culture.
The term xe2x80x9cfetal fibroblast cellxe2x80x9d as used herein refers to any differentiated fetal cell having a fibroblast appearance. While fibroblasts characteristically have a flattened appearance when cultured on culture media plates, fetal fibroblast cells can also have a spindle-like morphology. Fetal fibroblasts may require density limitation for growth, may generate type I collagen, and may have a finite life span in culture of approximately fifty generations. Preferably, fetal fibroblast cells rigidly maintain a diploid chromosomal content. For a description of fibroblast cells, see, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc., incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The terms xe2x80x9cmorphologyxe2x80x9d and xe2x80x9ccell morphologyxe2x80x9d as used herein refer to form, structure, and physical characteristics of cells. For example, one cell morphology is significant levels of alkaline phosphatase, and this cell morphology can be identified by determining whether a cell stains appreciably for alkaline phosphatase. Another example of a cell morphology is whether a cell is flat or round in appearance when cultured on a surface or in the presence of a layer of feeder cells. Many other cell morphologies are known to a person of ordinary skill in the art and are cell morphologies are readily identifiable using materials and methods well known to those skilled in the art. See, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.
The term xe2x80x9covarian follicular cellxe2x80x9d as used herein refers to a cultured or non-cultured cell obtained from an ovarian follicle, other than an oocyte. Follicular cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles. Furthermore, follicular cells may be isolated when an oocyte in an ovarian follicle is immature (i.e., an oocyte that has not progressed to metaphase II) or when an oocyte in an ovarian follicle is mature (i.e., an oocyte that has progressed to metaphase II or a later stage of development). Preferred follicular cells include, but are not limited to, pregranulosa cells, granulosa cells, theca cells, columnar cells, stroma cells, theca interna cells, theca extema cells, mural granulosa cells, luteal cells, and corona radiata cells. Particularly preferred follicular cells are cumulus cells. Various types of follicular cells are known and can be readily distinguished by those skilled in the art. See, e.g., Laboratory Production of Cattle Embryos, 1994, Ian Gordon, CAB International; Anatomy and Physiology of Farm Animals (5th ed.), 1992, R. D. Frandson and T. L. Spurgeon, Lea and Febiger, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables. Individual types of follicular cells may be cultured separately, or a mixture of types may be cultured together.
The termt xe2x80x9ccumulus cellxe2x80x9d as used herein refers to any cultured or non-cultured cell isolated from cells and/or tissue surrounding an oocyte. Persons skilled in the art can readily identify cumulus cells. Examples of methods for isolating and/or culturing cumulus cells are discussed in Damiani et al., 1996, Mol. Reprod. Dev. 45: 521-534; Long et al., 1994, J. Reprod. Fert. 102: 361-369; and Wakayama et al., 1998, Nature 394: 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings. Cumulus cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles. Cumulus cells may be isolated from oocytes in a number of manners well known to a person of ordinary skill in the art. For example, cumulus cells can be separated from oocytes by pipeting the cumulus cell/oocyte complex through a small bore pipette, by exposure to hyaluronidase, or by mechanically disrupting (e.g. vortexing) the cumulus cell/oocyte complex. Additionally, exposure to Ca++/Mg++ free media can remove cumulus from immature oocytes. Also, cumulus cell cultures can be established by placing matured oocytes in cell culture media. Once cumulus cells are removed from media containing increased LH/FSH concentrations, they can to attach to the culture plate.
The term xe2x80x9chepatic cellxe2x80x9d as used herein refers to any cultured or non-cultured cell isolated from a liver. Particularly preferred hepatic cells include, but are not limited to, a hepatic parenchymal cell, a Kuipffer cell, an Ito cell, a hepatocyte, a fat-storing cell, a pit cell, and a hepatic endothelial cell. Persons skilled in the art can readily identify the various types of hepatic cells. See, e.g., Regulation of Hepatic Metabolism, 1986, Thurman et al. (eds.), Plenum Press, which is incorporated herein by reference in its entirety including all figures, drawings, and tables.
The term xe2x80x9cdifferentiated cellxe2x80x9d as used herein refers to a precursor cell that has developed from an unspecialized phenotype to that of a specialized phenotype. For example, embryonic cells can differentiate into an epithelial cell lining the intestine. It is highly unlikely that differentiated cells revert into their precursor cells in vivo or in vitro. However, materials and methods of the invention can reprogram differentiated cells into immortalized, totipotent cells. Differentiated cells can be isolated from a fetus or a live born animal, for example.
In contrast to the totipotent and/or immortalized cells of the invention that arise from non-embryonic cells, an example of embryonic cells is discussed in WO 96/07732, entitled xe2x80x9cTotipotent Cells for Nuclear Transfer,xe2x80x9d hereby incorporated herein by reference in its entirety including all figures, drawings, and tables. The WO 96/07732 publication relates primarily to ovine animals. A unique feature of the present invention is that immortalized, totipotent cells are reprogrammed from non-embryonic cells by utilizing the materials and methods described herein in descriptions of the preferred embodiments and exemplary embodiments.
The term xe2x80x9casynchronous populationxe2x80x9d as used herein refers to cells that are not arrested at any one stage of the cell cycle. Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time. Some known stages of the cell cycle are G0, G1, S, G2, and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases. Cells can be arrested in the G0 stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by serum deprivation. Examples of methods for arresting non-immortalized cells in one part of the cell cycle are discussed in WO 97/07669, entitled xe2x80x9cQuiescent Cell Populations for Nuclear Transfer,xe2x80x9d hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The terms xe2x80x9csynchronous populationxe2x80x9d and xe2x80x9csynchronizingxe2x80x9d as used herein refer to a fraction of cells in a population that are arrested (i.e., the cells are not dividing) in a discreet ""stage of the cell cycle. Synchronizing a population of cells, by techniques such as serum deprivation, may render the cells quiescent. The term xe2x80x9cquiescentxe2x80x9d is defined below. Preferably, about 50% of the cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70% of the cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of the cells in a population of cells are arrested in one stage of the cell cycle. Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art. For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art. Alternatively, cells can be distinguished by size utilizing techniques well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example.
The terms xe2x80x9cserum deprivation,xe2x80x9d xe2x80x9cserum starved,xe2x80x9d and xe2x80x9cserum starvationxe2x80x9d as used herein refer to culturing cells in a medium comprising a serum concentration sufficiently low as to render cultured cells quiescent. The term xe2x80x9cquiescentxe2x80x9d is defined hereafter. A number of sera are used by those skilled in the art to supplement cell culture media. Particularly preferred is fetal bovine serum. Preferred serum starvation conditions are culturing cells in a medium comprising less than 1% fetal bovine serum. Particularly preferred conditions are culturing cells in a medium comprising not more than 0.5% fetal bovine serum. A length of time cultured cells are serum starved to be rendered quiescent can vary depending upon cell type. Cultured cells can be serum starved for at least 1 hour, at least 5 hours, at least 12 hours, and at least 24 hours. Preferably, cultured cells are serum starved for more than 1 day. Most preferably, cultured cells are serum starved for more than 3 days. These conditions are not meant to be limiting, and other serum starvation conditions can easily be identified by those skilled in the art without undue experimentation.
The term xe2x80x9cquiescentxe2x80x9d as used herein in reference to cells refers to cells which are not dividing. A xe2x80x9cquiescent cell culturexe2x80x9d refers to a culture in which a majority of cells in the culture are not dividing. More preferably, in a quiescent cell culture all cells in the culture are not dividing. As discussed herein, a cell culture may be rendered quiescent by serum starvation, but other methods which render cell cultures quiescent are known to those of ordinary skill in the art. Cells may be made permanently quiescent, and more preferably, quiescent cells may be made to resume dividing at a later time.
In preferred embodiments, (1) the totipotent cells of the invention comprise modified nuclear DNA; (2) the modified nuclear DNA includes a DNA sequence that encodes a recombinant product; (3) the recombinant product is a polypeptide; (4) the recombinant product is a ribozyme; (4) the recombinant product is expressed in a biological fluid or tissue; (5) the recombinant product confers or partially confers resistance to one or more diseases; (6) the recombinant product confers resistance or partially confers resistance to one or more parasites; (7) the modified nuclear DNA comprises at least one other DNA sequence that can function as a regulatory element; (8) the regulatory element is selected from the group consisting of promotor, enhancer, insulator, and repressor; and (9) the regulatory element is selected from the group consisting of milk protein promoter, urine protein promoter, blood protein promoter, tear duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, xcex2-casein promoter, melanocortin promoter, milk serum protein promoter, xcex1-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, xcex1-actin promoter.
The term xe2x80x9cmodified nuclear DNAxe2x80x9d as used herein refers to the nuclear deoxyribonucleic acid sequence of a cell, embryo, fetus, or animal of the invention that has been manipulated by one or more recombinant DNA techniques. Examples of these recombinant DNA techniques are well known to a person of ordinary skill in the art, which can include (1) inserting a DNA sequence from another organism (e.g., a human organism) into target nuclear DNA, (2) deleting one or more DNA sequences from target nuclear DNA, and (3) introducing one or more base mutations (e.g., site-directed mutations) into target nuclear DNA. Cells with modified nuclear DNA can be referred to as xe2x80x9ctransgenic cellsxe2x80x9d for the purposes of the invention. Transgenic cells can be useful as materials for nuclear transfer cloning techniques provided herein.
Methods and tools for insertion, deletion, and mutation of nuclear DNA of mammalian cells are well-known to a person of ordinary skill in the art. See, Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press; U.S. Pat. No. 5,633,067, xe2x80x9cMethod of Producing a Transgenic Bovine or Transgenic Bovine Embryo,xe2x80x9d DeBoer et al., issued May 27, 1997; U.S. Pat. No. 5,612,205, xe2x80x9cHomologous Recombination in Mammalian Cells,xe2x80x9d Kay et al., issued Mar. 18, 1997; and PCT publication WO 93/22432, xe2x80x9cMethod for Identifying Transgenic Pre-Implantation Embryos,xe2x80x9d all of which are incorporated by reference herein in their entirety, including all figures, drawings, and tables. These methods include techniques for transfecting cells with foreign DNA fragments and the proper design of the foreign DNA fragments such that they effect insertion, deletion, and/or mutation of the target DNA genome.
Transgenic cells may be obtained in a variety of manners. For example, transgenic cells can be isolated from a transgenic animal. Examples of transgenic animals are well known in the art, as described herein with regard to transgenic bovine and ovine animals. Cells isolated from a transgenic animal can be converted into totipotent and/or immortalized cells by using the materials and methods provided herein. In another example, transgenic cells can be created from totipotent and/or immortalized cells of the invention. Materials and methods for converting non-transgenic cells into transgenic cells are well known in the art, as described previously.
Any of the cell types defined herein can be altered to harbor modified nuclear DNA. For example, embryonic stem cells, fetal cells, and any totipotent and immortalized cell defined herein can be altered to harbor modified nuclear DNA.
Examples of methods for modifying a target DNA genome by insertion, deletion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, homologous recombination, gene targeting, transposable elements, and/or any other method for introducing foreign DNA. Other modification techniques well known to a person of ordinary skill in the art include deleting DNA sequences from a genome, and/or altering nuclear DNA sequences. Examples of techniques for altering nuclear DNA sequences are site-directed mutagenesis and polymerase chain reaction procedures. Therefore, the invention provides for bovine cells that are simultaneously totipotent, immortalized, and transgenic. These transgenic, totipotent, immortalized cells can serve as nearly unlimited sources of donor cells for production of cloned transgenic animals.
The term xe2x80x9crecombinant productxe2x80x9d as used herein refers to the product produced from a DNA sequence that comprises at least a portion of the modified nuclear DNA. This product can be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example. These products are well defined in the art. This list of products is for illustrative purposes only and the invention relates to other types of products.
The term xe2x80x9cribozymexe2x80x9d as used herein refers to ribonucleic acid molecules that can cleave other RNA molecules in specific-regions. Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region within that binding region or adjacent to the binding region. Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules. For specific descriptions of ribozymes, see U.S. Pat. No. 5,354,855, entitled xe2x80x9cRNA Ribozyme which Cleaves Substrate RNA without Formation of a Covalent Bond,xe2x80x9d Cech et al., issued on Oct. 11, 1994, and U.S. Pat. No. 5,591,610, entitled xe2x80x9cRNA Ribozyme Polymerases, Dephosphorylases, Restriction Endoribonucleases and Methods,xe2x80x9d Cech et al., issued on Jan. 7, 1997, both of which are incorporated by reference in their entireties including all figures, tables, and drawings.
The term xe2x80x9cbiological fluid or tissuexe2x80x9d as used herein refers to any fluid or tissue in a biological organism. The fluids may include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, oviductal fluid, and synovial fluid. The tissues may include, but are not limited to, lung, heart, blood, liver, muscle, brain, pancreas, skin, and others.
The term xe2x80x9cconfers resistancexe2x80x9d as used herein refers to the ability of a recombinant product to completely abrogate or partially alleviate the symptoms of a disease or parasitic condition. Hence, if the disease is related to inflammation, for example, a recombinant product can confer resistance to that inflammation if the inflammation decreases upon expression of the recombinant product. A recombinant product may confer resistance or partially confer resistance to a disease or parasitic condition, for example, if the recombinant product is an anti-sense RNA molecule that specifically binds to an mRNA molecule encoding a polypeptide responsible for the inflammation. Other examples of conferring resistance to diseases or parasites are described hereafter. In addition, examples of diseases are described hereafter.
Examples of parasites and strategies for conferring resistance to these parasites are described hereafter. These examples include, but are not limited to, worms, insects, invertebrate, bacterial, viral, and eukaryotic parasites. These parasites can lead to diseased states that can be controlled by the materials and methods of the invention.
The term xe2x80x9cregulatory elementxe2x80x9d as used herein refers to a DNA sequence that can increase or decrease the amount of product produced from another DNA sequence. The regulatory element can cause the constitutive production of the product (e.g., the product can be expressed constantly). Alternatively, the regulatory element can enhance or diminish the production of a recombinant product in an inducible fashion (e.g., the product can be expressed in response to a specific signal). The regulatory element can be controlled, for example, by nutrition, by light, or by adding a substance to the transgenic organism""s system. Examples of regulatory elements well-known to those of ordinary skill in the art are promoters, enhancers, insulators, and repressors. See, e.g., Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings.
The term xe2x80x9cpromotersxe2x80x9d or xe2x80x9cpromoterxe2x80x9d as used herein refers to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably operatively linked to the adjacent DNA sequence. A promoter typically increases the amount of recombinant product expressed from a DNA sequence as compared to the amount of the expressed recombinant product when no promoter exists. A promoter from one organism can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism. In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art. Examples of promoter elements are described hereafter.
The term xe2x80x9cenhancersxe2x80x9d or xe2x80x9cenhancerxe2x80x9d as used herein refers to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of the coding DNA sequence (e.g., the DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of the DNA sequence that encodes the recombinant product. Enhancer elements can increase the amount of recombinant product expressed from a DNA sequence above the increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.
The term xe2x80x9cinsulatorsxe2x80x9d or xe2x80x9cinsulatorxe2x80x9d as used herein refers to DNA sequences that flank the DNA sequence encoding the recombinant product. Insulator elements can direct the recombinant product expression to specific tissues in an organism. Multiple insulator elements are well known to persons of ordinary skill in the art. See, e.g., Geyer, 1997, Curr. Opin. Genet. Dev. 7:242-248, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The term xe2x80x9crepressorxe2x80x9d or xe2x80x9crepressor elementxe2x80x9d as used herein refers to a DNA sequence located in proximity to the DNA sequence that encodes the recombinant product, where the repressor sequence can decrease the amount of recombinant product expressed from that DNA sequence. Repressor elements can be controlled by the binding of a specific molecule or specific molecules to the repressor element DNA sequence. These molecules can either activate or deactivate the repressor element. Multiple repressor elements are available to a person of ordinary skill in the art.
The terms xe2x80x9cmilk protein promoter,xe2x80x9d xe2x80x9curine protein promoter,xe2x80x9d xe2x80x9cblood protein promoter,xe2x80x9d xe2x80x9ctear duct protein promoter,xe2x80x9d xe2x80x9csynovial protein promoter,xe2x80x9d and xe2x80x9cmandibular gland protein promoterxe2x80x9d refer to promoter elements that regulate the specific expression of proteins within the specified fluid or gland or cell type in an animal. For example, a milk protein promoter is a regulatory element that can control the expression of a protein that is expressed in the milk of an animal. Other promoters, such as casein promoter, xcex1-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and xcex1-actin promoter, for example, are well known to a person of ordinary skill in the art.
In preferred embodiments, (1) the totipotent cell is subject to manipulation; (2) the manipulation comprises the step of utilizing a totipotent cell in a nuclear transfer procedure; (3) the manipulation comprises the step of cryopreserving the totipotent cells; (4) the manipulation comprises the step of thawing the totipotent cells; (5) the manipulation comprises the step of passaging totipotent cells; (6) the manipulation comprises the step of synchronizing totipotent cells; (7) the manipulation comprises the step of transfecting totipotent cells with foreign DNA; and (8) the manipulation comprises the step of dissociating a cell from another cell or group of cells.
The term xe2x80x9cmanipulationxe2x80x9d as used herein refers to the common usage of the term, which is the management or handling directed towards some object. Examples of manipulations are described herein.
The term xe2x80x9cnuclear transferxe2x80x9d as used herein refers to introducing a fall complement of nuclear DNA from one cell to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384, entitled xe2x80x9cMultiplying Bovine Embryos,xe2x80x9d Prather et al., issued on Feb. 19, 1991 and U.S. Pat. No. 5,057,420, entitled xe2x80x9cBovine Nuclear Transplantation,xe2x80x9d Massey, issued on Oct. 15, 1991, both of which are hereby incorporated by reference in their entirety including all figures, tables and drawings. Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.
Although the basic principals of nuclear transfer have been described previously, the technique can be sensitive to the introduction of any new parameters. Therefore, significant modifications to the techniques described in the area of nuclear transfer may require some experimentation to determine the practical effect of these modifications upon the efficiency of nuclear transfer. An example of a variable that can affect nuclear transfer efficiency is the age of the oocyte utilized for enucleation and nuclear transfer.
The term xe2x80x9ccryopreservingxe2x80x9d as used herein refers to freezing a cell, embryo, or animal of the invention. The cells, embryos, or portions of animals of the invention are frozen at temperatures preferably lower than 0xc2x0 C., more preferably lower than xe2x88x9280xc2x0 C., and most preferably at temperatures lower than xe2x88x92196xc2x0 C. Cells and embryos in the invention can be cryopreserved for an indefinite amount of time. It is known that biological materials can be cryopreserved for more than fit years. For example, semen that is cryopreserved for more than fifty years can be utilized to artificially inseminate a female bovine animal. Methods and tools for cryopreservation are well-known to those skilled in the art. See, e.g., U.S. Pat. No. 5,160,312, entitled xe2x80x9cCryopreservation Process for Direct Transfer of Embryos,xe2x80x9d issued to Voelkel on Nov. 3, 1992.
The term xe2x80x9cthawingxe2x80x9d as used herein refers to the process of increasing the temperature of a cryopreserved cell, embryo, or portions of animals. Methods of thawing cryopreserved materials such that they are active after the thawing process are well-known to those of ordinary skill in the art.
The terms xe2x80x9ctransfected,xe2x80x9d xe2x80x9ctransformation,xe2x80x9d and xe2x80x9ctransfectionxe2x80x9d as used herein refer to methods of inserting foreign DNA into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, liposomes, polycationic micelles, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest. Transfection techniques are well known to a person of ordinary skill in the art and materials and methods for carrying out transfection of DNA constructs into cells are commercially available. Materials typically used to transfect cells with DNA constructs are lipophilic compounds such as Lipofectin(trademark). Particular lipophilic compounds can be induced to form liposomes for mediating transfection of the DNA construct into the cells. These specified methods are not limiting and the invention relates to any transformation technique well known to a person of ordinary skill in the art. See, e.g., Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press and Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, both of which were previously incorporated by reference.
The term xe2x80x9cforeign DNAxe2x80x9d as used herein refers to DNA that can be transfected into a target cell, where the foreign DNA harbors at least one base pair modification as compared to the nuclear DNA of the target organism. Foreign DNA and transfection can be further understood and defined in conjunction with the term xe2x80x9cmodified nuclear DNA,xe2x80x9d described previously.
The term xe2x80x9cdissociatingxe2x80x9d as used herein refers to the materials and methods useful for pulling a cell away from another cell. For example, a blastomere (i.e., a cellular member of a blastocyst stage embryo) can be pulled away from the rest of the developing cell mass by techniques and apparatus well known to a person of ordinary skill in the art. See, e.g., U.S. Pat. No. 4,994,384, entitled xe2x80x9cMultiplying Bovine Embryos,xe2x80x9d issued on Feb. 19, 1991, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. Alternatively, cells proliferating in culture can be separated from one another to facilitate such processes as cell passaging, which is described previously. In addition, dissociation of a cultured cell from a group of cultured cells can be useful as a first step in the process of nuclear transfer, as described hereafter. When a cell is dissociated from an embryo, the dissociation manipulation can be useful for such processes as re-cloning, a process described herein, as well as a step for multiplying the number of embryos.
In another aspect, the invention features a totipotent mammalian cell, where the cell is immortalized, prepared by a process comprising the steps of: (a) isolating at least one precursor cell; and (b) introducing a stimulus to the precursor cell that converts the precursor cell into the totipotent mammalian cell.
The term xe2x80x9cconvertsxe2x80x9d as used herein refers to the phenomenon in which precursor cells become immortalized and/or totipotent. The term xe2x80x9cconvertxe2x80x9d is synonymous with the term xe2x80x9creprogramxe2x80x9d as used herein when the precursor cell is non-immortalized and/or non-totipotent. Precursor cells can be converted into totipotent, immortalized cells in varying proportions. For example, it is possible that only a small portion of precursor cells are converted into totipotent, immortalized cells. In the art, some researchers have discussed techniques for converting precursor cells into pluripotent cells. Matsui et al., 1992, Cell 70: 841-847.
The term xe2x80x9cstimulusxe2x80x9d as used herein refers to materials and/or methods useful for converting precursor cells into immortalized and/or totipotent cells. The stimulus can be electrical, mechanical, temperature-related, and/or chemical, for example. The stimulus may be a combination of one or more different types of stimuli. As described herein in exemplary embodiments, placing precursor cells in culture can be a sufficient stimulus to convert precursor cells into immortalized and/or totipotent cells. A stimulus can be introduced to precursor cells for any period of time that accomplishes the conversion of precursor cells into immortalized and/or totipotent cells.
The term xe2x80x9cintroducexe2x80x9d as used herein in reference to a stimulus refers to a step or steps in which precursor cells are contacted with a stimulus. If the stimulus is chemical in nature, for example, the stimulus may be introduced to the precursor cells by mixing the stimulus with cell culture medium.
In preferred embodiments (1) the precursor cells are co-cultured with feeder cells; (2) the precursor cells are not co-cultured with feeder cells; (3) the feeder cells are established from fetal cells; (4) the fetal cells arise from a fetus where no cell types have been removed from the fetus; (5) the fetal cells arise from a fetus where one or more cell types have been removed from the fetus; (6) the stimulus is introduced to precursor cells by feeder cells; (7) the feeder cells are the only source of the stimulus; (8) the stimulus is introduced to the precursor cells in a mechanical fashion; (9) the only stimulus is introduced to the precursor cells in a mechanical fashion; (10) the stimulus is introduced to the precursor cells by feeder cells and in a mechanical fashion; (11) the stimulus comprises the step of incubating the precursor cells with a receptor ligand cocktail; (12) the precursor cells are isolated from an ungulate animal and preferably a bovine animal; (13) the precursor cells are selected from the group consisting of non-embryonic cells, primordial germ cells, genital ridge cells, amniotic cells, fetal fibroblast cells, ovarian follicular cells, cumulus cells, hepatic cells, differentiated cells, cells that originate from an animal, embryonic stem cells, fetal cells, and embryonic cells; (14) the receptor ligand cocktail comprises at least one component selected from the group consisting of cytokine, growth factor, trophic factor, and neurotrophic factor, LIF, and FGF; (15) the LIF has an amino acid sequence substantially similar to the amino acid sequence of human LIF; and (16) the FGF has an amino acid sequence substantially similar to the amino acid sequence of bovine bFGF.
The terms xe2x80x9cmechanical fashionxe2x80x9d and xe2x80x9cmechanical stimulusxe2x80x9d as used herein refers to introducing a stimulus to cells where the stimulus is not introduced by feeder cells. For example, purified LIF and bFGF (defined hereafter) can be introduced as a stimulus to precursor cells by adding these purified products to a cell culture medium in which the precursor cells are growing.
The term xe2x80x9cfeeder cellsxe2x80x9d as used herein refers to cells grown in co-culture with target cells. Target cells can be precursor cells and totipotent cells, for example. Feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines such as LIF and steel factor), and metabolic nutrients to target cells. Certain cells, such as immortalized, totipotent cells may not require feeder cells for healthy growth. Feeder cells preferably grow in a mono-layer.
Feeder cells can be established from multiple cell types. Examples of these cell types are fetal cells, mouse cells, Buffalo rat liver cells, and oviductal cells. These examples are not meant to be limiting. Tissue samples can be broken down to establish a feeder cell line by methods well known in the art (e.g., by using a blender). Feeder cells may originate from the same or different animal species as the precursor cells. In an example of feeder cells established from fetal cells, ungulate fetuses and preferably bovine fetuses may be utilized to establish a feeder cell line where one or more cell types have been removed from the fetus (e.g., primordial germs cells, cells in the head region, and cells in the body cavity region). When an entire fetus is utilized to establish a fetal feeder cell line, feeder cells (e.g., fibroblast cells) and precursor cells (e.g., primordial germ cells) can arise from the same source (e.g., one fetus).
The term xe2x80x9creceptor ligand cocktailxe2x80x9d as used herein refers to a mixture of one or more receptor ligands. A receptor ligand refers to any molecule that binds to a receptor protein located on the outside or the inside of a cell. Receptor ligands can be selected from molecules of the cytokine family of ligands, neurotrophin family of ligands, growth factor family of ligands, and mitogen family of ligands, all of which are well known to a person of ordinary skill in the art. Examples of receptor/ligand pairs are: epidermal growth factor receptor/epidermal growth factor, insulin/insulin receptor, cAMP-dependent protein kinase/cAMP, growth hormone receptor/growth hormone, and steroid receptor/steroid. It has been shown that certain receptors exhibit cross-reactivity. For example, heterologous receptors, such as insulin-like growth factor receptor 1 (IGFR1) and insulin-like growth factor receptor 2 (IGFR2) can both bind IGF1. When a receptor ligand cocktail comprises the stimulus, the receptor ligand cocktail can be introduced to the precursor cell in a variety of manners known to a person of ordinary skill in the art.
The term xe2x80x9ccytokinexe2x80x9d as used herein refers to a large family of receptor ligands well-known to a person of ordinary skill in the art. The cytokine family of receptor ligands includes such members as leukemia inhibitor factor (LIF), cardiotrophin 1 (CT-1), ciliary neurotrophic factor (CNTF), stem cell factor (SCF), oncostatin M (OSM), and any member of the interleukin (IL) family, including IL-6, IL-1, and IL-12. The teachings of the invention do not require the mechanical addition of steel factor (also known as stem cell factor in the art) for the conversion of precursor cells into totipotent cells.
The term xe2x80x9cgrowth factorxe2x80x9d as used herein refers to any receptor ligand that causes a cell growth and/or cell proliferation effect. Examples of growth factors are well known in the art. Fibroblast growth factor (FGF) is one example of a growth factor. The term xe2x80x9cbFGFxe2x80x9d can refer to basic FGF.
The term xe2x80x9csubstantially similarxe2x80x9d as used herein in reference to amino acid sequences refers to two amino acid sequences having preferably 50% or more amino acid identity, more preferably 70% or more amino acid identity or most preferably 90% or more amino acid identity. Amino acid identity is a property of amino acid sequence that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those of ordinary skill in the art will recognize that several computer programs are available for performing sequence comparisons and determining sequence identity.
In another aspect, the invention features a method for preparing a totipotent mammalian cell, where the cell is immortalized, comprising the following steps: (a) isolating one or more precursor cells; and (b) introducing the precursor cell to a stimulus that converts the precursor cell into the totipotent cell. Any of the embodiments defined previously herein in reference to totipotent mammalian cells relate to the method for preparing a totipotent mammalian cell.
The invention relates in part to cloned totipotent embryos. Hence, aspects of the invention feature cloned mammalian embryos where (1) the embryo is totipotent; (2) the embryo arises from an immortalized and/or totipotent cell; and (3) the embryo arises from a non-embryonic cell; and (4) any combination of the foregoing.
The term xe2x80x9ctotipotentxe2x80x9d as used herein in reference to embryos refers to embryos that can develop into a live born animal. The term xe2x80x9clive bornxe2x80x9d is defined previously.
The term xe2x80x9cclonedxe2x80x9d as used herein refers to a cell, embryonic cell, fetal cell, and/or animal cell having a nuclear DNA sequence that is substantially similar or identical to the nuclear DNA sequence of another cell, embryonic cell, fetal cell, and/or animal cell. The terms xe2x80x9csubstantially similarxe2x80x9d and xe2x80x9cidenticalxe2x80x9d are described herein. The cloned embryo can arise from one nuclear transfer, or alternatively, the cloned embryo can arise from a cloning process that includes at least one re-cloning step. If the cloned embryo arises from a cloning procedure that includes at least one re-cloning step, then the cloned embryo can indirectly arise from an immortalized, totipotent cell since the re-cloning step can utilize embryonic cells isolated from an embryo that arose from an immortalized, totipotent cell.
In preferred embodiments, (1) the cloned mammalian embryo is preferably an ungulate embryo and more preferably a bovine embryo; (2) the cloned bovine embryo can be one member of a plurality of embryos, where the plurality of embryos share a substantially similar nuclear DNA sequence; (3) the cloned mammalian embryo can be one member of a plurality of embryos, and the plurality of embryos can have an identical nuclear DNA sequence; (4) the cloned mammalian embryo has a nuclear DNA sequence that is substantially similar to a nuclear DNA sequence of a live born mammalian animal; (5) one or more cells of the cloned mammalian embryo have modified nuclear DNA; (6) the cloned mammalian embryo is subject to manipulation; (7) the manipulation comprises the step of culturing the embryo in a suitable medium; (8) the suitable medium for culturing the embryo is CR-2 medium; (9) the medium can comprise feeder cells; (10) the manipulation of an embryo comprises the step of implanting the embryo into the uterus of a female; (11) the female animal is preferably an ungulate animal and more preferably a bovine animal; (12) the estrus cycle of the female is synchronized with the development cycle of the embryo; and (13) the manipulation comprises the step of incubating the embryo in an artificial environment.
All preferred embodiments related to modified nuclear DNA for totipotent cells of the invention extend to cloned embryos of the invention. In addition, any of the manipulations described in conjunction with totipotent cells of the invention apply to cloned embryos of the invention.
The term xe2x80x9csubstantially similarxe2x80x9d as used herein in reference to nuclear DNA sequences refers to two nuclear DNA sequences that are nearly identical. The two sequences may differ by copy error differences that normally occur during the replication of a nuclear DNA. Substantially similar DNA sequences are preferably greater than 97% identical, more preferably greater than 98% identical, and most preferably greater than 99% identical. The term xe2x80x9cidentityxe2x80x9d as used herein in reference to nuclear DNA sequences can refer to the same usage of the term in reference to amino acid sequences, which is described previously herein.
The term xe2x80x9cpluralityxe2x80x9d as used herein in reference to embryos refers to a set comprising at least two embryos having a substantially similar nuclear DNA sequence. In preferred embodiments, the plurality consists of five or more embryos, ten or more embryos, one-hundred or more embryos, or one-thousand or more embryos. Because the occurrence of more than three embryos progressing to term only occurs with a probability of approximately 1/100,000, a plurality of at least five embryos or animals relates to cloned embryos or cloned animals rather than naturally occurring embryos or animals.
The term xe2x80x9cculturingxe2x80x9d as used herein with respect to embryos refers to laboratory procedures that involve placing an embryo in a culture medium. The embryo can be placed in the culture medium for an appropriate amount of time to allow the embryo to remain static but functional in the medium, or to allow the embryo to grow in the medium. Culture media suitable for culturing embryos are well-known to those skilled in the art. See, e.g., U.S. Pat. No. 5,213,979, entitled xe2x80x9cIn vitro Culture of Bovine Embryos,xe2x80x9d First et at., issued May 25, 1993, and U.S. Pat. No. 5,096,822, entitled xe2x80x9cBovine Embryo Medium,xe2x80x9d Rosenkrans, Jr. et al., issued Mar. 17, 1992, incorporated herein by reference in their entireties including all figures, tables, and drawings.
The term xe2x80x9csuitable mediumxe2x80x9d as used herein refers to any medium that allows cell proliferation. The suitable medium need not promote maximum proliferation, only measurable cell proliferation. A suitable medium for embryo development is discussed previously.
The term xe2x80x9cCR-2 mediumxe2x80x9d as used herein refers to a medium suitable for culturing embryos. CR-2 medium can comprise one or more of the following components: sodium chloride; potassium chloride; sodium bicarbonate; hemicalcium L-lactate; and fatty-acid free BSA. These components may exist in the medium in concentrations of about 115 mM for sodium chloride; about 3 mM for potassium chloride; about 25 mM for sodium bicarbonate; about 5 mM for hemicalcium L-lactate; and about 3 mg/mL for fatty-acid free BSA. Alternatively, the concentrations of these components may exist in the medium in concentrations of 0-1 M sodium chloride; 0-100 mM potassium chloride; 0-500 mM sodium bicarbonate; 0-100 mM hemicalcium L-lactate; and 0-100 mg/mL fatty-acid free BSA.
The term xe2x80x9cfeeder cellsxe2x80x9d is defined previously herein. Embryos of the invention can be cultured in media with or without feeder cells. In other preferred embodiments, the feeder cells can be cumulus cells.
The term xe2x80x9cimplantingxe2x80x9d as used herein in reference to embryos refers to impregnating a-female animal with an embryo described herein. This technique is well known to a person of ordinary skill in the art. See, e.g., Seidel and Elsden, 1997, Embryo Transfer in Dairy Cattle, W. D. Hoard and Sons, Co., Hoards Dairyman. The embryo may be allowed to develop in utero, or alternatively, the fetus may be removed from the uterine environment before parturition.
The term xe2x80x9csynchronizedxe2x80x9d as used herein in reference to estrus cycle, refers to assisted reproductive techniques well known to a person of ordinary skill in the art. These techniques are fully described in the reference cited in the previous paragraph. Typically, estrogen and progesterone hormones are utilized to synchronize the estrus cycle of the female animal with the developmental cycle of the embryo. The term xe2x80x9cdevelopmental cyclexe2x80x9d as used herein refers to embryos of the invention and the time period that exists between each cell division within the embryo. This time period is predictable for embryos from ungulates, and can be synchronized with the estrus cycle of a recipient animal.
The term xe2x80x9cartificial environmentxe2x80x9d refers to one that promotes the development of an embryo or other developing cell mass. An artificial environment can be a uterine environment or an oviductal environment of a species different from that of the developing cell mass. For example, a developing bovine embryo can be placed into the uterus or oviduct of an ovine animal. Stice and Keefer, 1993, xe2x80x9cMultiple generational bovine embryo cloning,xe2x80x9d Biology of Reproduction 48: 715-719. Alternatively, an artificial development environment can be assembled in vitro. This type of artificial uterine environment can be synthesized using biological and chemical components known in the art.
In another aspect the invention features a cloned mammalian embryo, where the embryo is totipotent, prepared by a process comprising the step of nuclear transfer. Preferably, nuclear transfer occurs between (a) a totipotent mammalian cell, where the cell is immortalized, and (b) an oocyte, where the oocyte is at a stage allowing formation of the embryo.
In preferred embodiments, (1) the oocyte is an enucleated oocyte; (2) the totipotent mammalian cell and the oocyte preferably originate from an ungulate animal and more preferably originate from a bovine animal; (3) the totipotent mammalian cell can originate from one specie of ungulate and the oocyte can originate from another specie of ungulate; (4) the oocyte is a young oocyte; (5) the totipotent mammalian cell is placed in the perivitelline space of the oocyte; (6) the totipotent cell utilized for nuclear transfer can arise from any of the cells described previously (e.g., a non-embryonic cell, a primordial germ cell, a genital ridge cell, a differentiated cell, a fetal cell, a non-fetal cell, a non-primordial germ cell, an amniotic cell, a fetal fibroblast cell, an ovarian follicular cell, a cumulus cell, an hepatic cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells, a cell isolated from an existing animal, and an embryonic stem cell); (7) the nuclear transfer comprises the step of translocation of the totipotent mammalian cell into the recipient oocyte; (8) the translocation can comprise the step of injection of the totipotent mammalian cell nuclear donor into the recipient oocyte; (9) the translocation can comprise the step of fusion of the totipotent mammalian cell and the oocyte; (10) the fusion can comprise the step of delivering one or more electrical pulses to the totipotent mammalian cell and the oocyte; (11) the fusion can comprise the step of delivering a suitable concentration of at least one fusion agent to the totipotent mammalian cell and the oocyte; (12) the nuclear transfer may comprise the step of activation of the totipotent mammalian cell and the oocyte; and (13) the activation is accomplished by introducing DMAP and/or ionomycin to an oocyte and/or a cybrid.
The term xe2x80x9cenucleated oocytexe2x80x9d as used herein refers to an oocyte which has had part of its contents removed. Typically a needle can be placed into an oocyte and the nucleus can be aspirated into the inner space of the needle. The needle can be removed from the oocyte without rupturing the plasma membrane. This enucleation technique is well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420; and Willadsen, 1986, Nature 320:63-65. An enucleated oocyte can be prepared from a young or an aged oocyte. Definitions of xe2x80x9cyoung oocytexe2x80x9d and xe2x80x9caged oocytexe2x80x9d are provided herein; Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enuicleated oocytes, and the cytoplasm of one or more enucleated oocytes.
The term xe2x80x9ccybridxe2x80x9d as used herein refers to a construction where an entire nuclear donor is translocated into the cytoplasm of a recipient oocyte. See, e.g., In Vitro Cell. Dev. Biol. 26: 97-101 (1990).
The invention specifically relates to cloned mammalian embryos created by nuclear transfer, where the nucleus of the oocyte is not physically extracted from the nucleus. It is possible to create a cloned embryo where the nuclear DNA from the donor cell is the material replicated during cellular divisions. See, e.g. Wagoner et al., 1996, xe2x80x9cFunctional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,xe2x80x9d Theriogenology 46: 279-284.
The term xe2x80x9canother ungulatexe2x80x9d as used herein refers to a situation where the nuclear donor originates from an ungulate of a different species, genera or family than the ungulate from which the recipient oocyte originates. For example, the totipotent mammalian cell used as a nuclear donor can arise from a water buffalo, while the oocyte recipient can arise from a domestic cow. This example is not meant to be limiting and any ungulate species/family combination of nuclear donors and recipient oocytes are foreseen by the invention.
The term xe2x80x9cyoung oocytexe2x80x9d as used herein refers to an oocyte that has been matured in vitro and/or ovulated in vivo for less than 28 hours since the onset of maturation. Oocytes can be isolated from live animals using methods well known to a person of ordinary skill in the art. See, e.g., Pieterse et al., 1988, xe2x80x9cAspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries,xe2x80x9d Theriogenology 30: 751-762. Oocytes can be isolated from ovaries or oviducts or deceased or live born animals. Suitable media for in vitro culture of oocytes are well known to a person of ordinary skill in the art. See, e.g., U.S. Pat. No. 5,057,420, which is incorporated by reference herein.
The term xe2x80x9cmaturationxe2x80x9d as used herein refers to process in which an oocyte is incubated in a medium in vitro. Oocytes can be incubated with multiple media well known to a person of ordinary skill in the art. See, e.g., Saito et al., 1992, Roux""s Arch. Dev. Biol. 201: 134-141 for bovine organisms and Wells et al., 1997, Biol. Repr. 57: 385-393 for ovine organisms, both of which are incorporated herein by reference in their entireties including all figures, tables, and drawings. Maturation media can comprise multiple types of components, including microtubule inhibitors (e.g., cytochalasin B). Other examples of components that can be incorporated into maturation media are discussed in WO 97/07668, entitled xe2x80x9cUnactivated Oocytes as Cytoplast Recipients for Nuclear Transfer,xe2x80x9d Campbell and Wilmut, published on Mar. 6, 1997, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. The time of maturation can be determined from the time that an oocyte is placed in a maturation medium and the time that the oocyte is then utilized in a nuclear transfer procedure.
Young oocytes can be identified by the appearance of their ooplasm.
Because certain cellular material (e.g., lipids) have not yet dispersed within the ooplasm. Young oocytes can have a pycnotic appearance. A pycnotic appearance can be characterized as clumping of cytoplasmic material. A xe2x80x9cpycnoticxe2x80x9d appearance is to be contrasted with the appearance of oocytes that are older than 28 hours, which have a more homogenous appearing ooplasm.
The term xe2x80x9ctranslocationxe2x80x9d as used herein in reference to nuclear transfer refers to the combining of a totipotent mammalian cell nuclear donor and a recipient oocyte. The translocation may be performed by such techniques as fusion and/or direct injection, for example.
The term xe2x80x9cinjectionxe2x80x9d as used herein in reference to embryos, refers to the perforation of the oocyte with a needle, and insertion of the nuclear donor in the needle into the oocyte. In preferred embodiments, the nuclear donor may be injected into the cytoplasm of the oocyte or in the perivitelline space of the oocyte. This direct injection approach is well known to a person of ordinary skill in the art, as indicated by the publications already incorporated herein in reference to nuclear transfer. For the direct injection approach to nuclear transfer, the whole totipotent mammalian cell may be injected into the oocyte, or alternatively, a nucleus isolated from the totipotent mammalian cell may be injected into the oocyte. Such an isolated nucleus may be surrounded by nuclear membrane only, or the isolated nucleus may be surrounded by nuclear membrane and plasma membrane in any proportion. The oocyte may be pre-treated to enhance the strength of its plasma membrane, such as by incubating the oocyte in sucrose prior to injection of the nuclear donor.
Techniques for placing a nuclear donor (e.g., an immortalized and totipotent cell of the invention) into the perivitelline space of an enucleated oocyte are well known to a person of ordinary skill in the art, and are fully described in the patents and references cited previously herein in reference to nuclear transfer.
The term xe2x80x9cfusionxe2x80x9d as used herein refers to the combination of portions of lipid membranes corresponding to the totipotent mammalian cell nuclear donor and the recipient oocyte. Lipid membranes can correspond to the plasma membranes of cells or nuclear membranes, for example. The fusion can occur between the nuclear donor and recipient oocyte when they are placed adjacent to one another, or when the nuclear donor is placed in the perivitelline space of the recipient oocyte, for example. Specific examples for translocation of the totipotent mammalian cell into the oocyte are described hereafter in other preferred embodiments. These techniques for translocation are fully described in the references cited previously herein in reference to nuclear transfer.
The term xe2x80x9celectrical pulsesxe2x80x9d as used herein refers to subjecting the nuclear donor and recipient oocyte to electric current. For nuclear transfer, the nuclear donor and recipient oocyte can be aligned between electrodes and subjected to electrical current. The electrical current can be alternating current or direct current. The electrical current can be delivered to cells for a variety of different times as one pulse or as multiple pulses. The cells are typically cultured in a suitable medium for the delivery of electrical pulses. Examples of electrical pulse conditions utilized for nuclear transfer are described in the references and patents previously cited herein in reference to nuclear transfer.
The term xe2x80x9cfusion agentxe2x80x9d as used herein refers to any compound or biological organism that can increase the probability that portions of plasma membranes from different cells will fuse when a totipotent mammalian cell nuclear donor is placed adjacent to the recipient oocyte. In preferred embodiments fusion agents are selected from the group consisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus. These examples are not meant to be limiting and other fusion agents known in the art are applicable and included herein.
The term xe2x80x9csuitable concentrationxe2x80x9d as used herein in reference to fusion agents, refers to any concentration of a fusion agent that affords a measurable amount of fusion. Fusion can be measured by multiple techniques well known to a person of ordinary skill in the art, such as by utilizing a light microscope, dyes, and fluorescent lipids, for example.
The term xe2x80x9cactivationxe2x80x9d refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step. Cybrids may require stimulation in order to divide after a nuclear transfer has occurred. The invention pertains to any activation materials and methods known to a person of ordinary skill in the art. Although electrical pulses are sometimes sufficient for stimulating activation of cybrids, other means are sometimes useful or necessary for proper activation of the cybrid. Chemical materials and methods usefull for activating embryos are described below in other preferred embodiments of the invention.
Examples of non-electrical means for activation include agents such as ethanol; inositol trisphosphate (IP3); Ca++ ionophores (e.g., ionomycin) and protein kinase inhibitors (e.g., 6-dimethylaminopurine (DMAP)); temperature change; protein synthesis inhibitors (e.g., cyclohexamide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanical techniques; and thapsigargin. The invention includes any activation techniques known in the art. See, e.g., U.S. Pat. No. 5,496,720, entitled xe2x80x9cParthenogenic Oocyte Activation,xe2x80x9d issued on Mar. 5, 1996, Susko-Parrish et al., incorporated by reference herein in its entirety, including all figures, tables, and drawings.
In other preferred embodiments, (1) one or more cells of the cloned embryo comprise modified nuclear DNA; (2) the cloned embryo is subject to manipulation; (3) the manipulation comprises the step of disaggregating at least one individual cell from a cloned embryo; (4) the manipulation comprises the step of utilizing the individual cell as a nuclear donor in a nuclear transfer procedure; (5) the individual cell is disaggregated from the inner cell mass of a blastocyst stage embryo; (6) the individual cell is disaggregated from a pre-blastocyst stage embryo; (7) the manipulation comprises the process of re-cloning; (8) the re-cloning process comprises the steps of: (a) separating the embryo into one or more individual cells, and (b) performing at least one subsequent nuclear transfer between (i) an individual cell of (a), and (ii) an oocyte; (9) the oocyte utilized for the subsequent nuclear transfer is an aged oocyte; (10) the individual cell is placed in the perivitelline space of the enucleated oocyte for the subsequent nuclear transfer; (11) the subsequent nuclear transfer comprises at least one of the steps of translocation, injection, fusion, and activation of the individual cell and/or the enucleated oocyte; (12) one or more cells of the cloned mammalian embryo arising from the subsequent nuclear transfer comprises modified nuclear DNA; and (13) the cloned mammalian embryo arising from the subsequent nuclear transfer may be subject to a subsequent manipulation, where the subsequent manipulation is any of the manipulation steps defined previously herein in relation to immortalized cells and/or cloned embryos.
The term xe2x80x9cindividual cellsxe2x80x9d as used herein refers to cells that have been isolated from a cloned mammalian embryo of the invention. An individual single cell can be isolated from the rest of the embryonic mass by techniques well known to those skilled in the art. See, U.S. Pat. Nos. 4,994,384 and 5,957,420, previously incorporated herein by reference in their entireties.
The term xe2x80x9csubsequent nuclear transferxe2x80x9d as described herein is also referred to as a xe2x80x9cre-cloningxe2x80x9d step. Preferably, a re-cloning step can be utilized to enhance nuclear reprogramming during nuclear transfer, such that the product of nuclear transfer is a live born animal. The re-cloning step is distinct, since previous efforts towards re-cloning have been directed to multiplying embryo number and not for enhancement of nuclear reprogramming. The number of subsequent nuclear transfer steps is discussed in greater detail hereafter.
Any of the preferred embodiments related to the translocation, injection, fusion, and activation steps described previously herein relate to the subsequent nuclear transfer step.
The term xe2x80x9cinner cell massxe2x80x9d as used herein refers to the cells that gives rise to the embryo proper. The cells that line the outside of a blastocyst are referred to as the trophoblast of the embryo. The methods for isolating inner cell mass cells from an embryo are well known to a person of ordinary skill in the art. See, Sims and First, 1993, Theriogenology 39:313; and Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268, hereby incorporated by reference herein in their entireties, including all figures, tables, and drawings. The term xe2x80x9cpre-blastocystxe2x80x9d is well known in the art and is referred to previously.
The term xe2x80x9caged oocytexe2x80x9d as used herein refers to an oocyte that has been matured in vitro or ovulated in vivo for more than 28 hours since the onset of maturation or ovulation. An aged oocyte can be identified by its characteristically homogenous ooplasm. This appearance is to be contrasted with the pycnotic appearance of young oocytes as described previously herein. The age of the oocyte can be defined by the time that has elapsed between the time that the oocyte is placed in a suitable maturation medium and the time that the oocyte is activated. The age of the oocyte can dramatically enhance the efficiency of nuclear transfer.
The term xe2x80x9covulated in vivoxe2x80x9d as used herein refers to an oocyte that is isolated from an animal a certain number of hours after the animal exhibits characteristics that it is in estrus. The characteristics of an animal in estrus are well known to a person of ordinary skill in the art, as described in references disclosed herein.
In another aspect the invention relates to a method for preparing a cloned mammalian embryo. The method comprises the step of a nuclear transfer between: (a) a totipotent mammalian cell, where the cell is immortalized; and (b) an oocyte, where the oocyte is at a stage allowing formation of the embryo. In preferred embodiments, any of the embodiments of the invention concerning cloned mammalian embryos apply to methods for preparing cloned mammalian embryos.
In another aspect, the invention features cloned mammalian fetuses arising from totipotent embryos of the invention. Preferably, the mammalian fetuses are ungulate fetuses, and more preferably, the ungulate fetuses are bovine fetuses. A fetus may be isolated from the uterus of a pregnant female animal.
In preferred embodiments, (1) one or more cells of the fetuses harbor modified nuclear DNA (defined previously herein); and (2) the fetuses may be subject to any of the manipulations defined herein. For example, one manipulation may comprise the steps of isolating a fetus from the uterus of a pregnant female animal, isolating a cell from the fetus (e.g., a primordial germ cell), and utilizing the isolated cell as a nuclear donor for nuclear transfer.
Other aspects of the invention feature (1) a cloned mammalian fetus prepared by a process comprising the steps of (a) preparation of a cloned mammalian embryo defined previously, and (b) manipulation of the cloned mammalian embryo such that it develops into a fetus; (2) a method for preparing a cloned mammalian fetus comprising the steps of (a) preparation of a cloned mammalian embryo defined previously, and (b) manipulation of the cloned mammalian embryo such that it develops into a fetus; (3) a method of using a cloned fetus of the invention comprising the step of isolating at least one cell type from a fetus (e.g., for creating a feeder cell layer); and (4) a method of using a cloned fetus of the invention comprising the step of separating at least one part of a fetus into individual cells (e.g., for establishing a feeder cell layer).
In another aspect the invention features a cloned mammalian animal arising from a cloned embryo of the invention. The embryo is totipotent and can arise from any of the processes or methods described previously herein.
In preferred embodiments, the cloned mammalian animal (1) is preferably a cloned ungulate animal and more preferably a cloned bovine animal; and (2) is equal in age or older than an animal selected from the group consisting of pre- and post-pubertal animals.
In yet another aspect the invention relates to a cloned mammalian animal, where the animal is one member of a plurality of animals, and where the plurality of animals have a substantially similar nuclear DNA sequence. The term xe2x80x9csubstantially similarxe2x80x9d in relation to nuclear DNA sequences is defined previously herein.
In preferred embodiments, (1) the plurality consists of five or more animals, ten or more animals, one-hundred or more animals, and one-thousand or more animals; and (2) the plurality of animals can have an identical nuclear DNA sequence. The term xe2x80x9cidenticalxe2x80x9d in reference to nuclear DNA sequences is described previously herein.
In another aspect, the invention relates to a cloned mammalian animal having one or more cells that comprise modified nuclear DNA. All of the preferred embodiments relating to modified nuclear DNA described previously apply to cloned bovine animals of the invention.
In yet another aspect, the invention features a method of using a cloned mammalian animal, comprising the step of isolating at least one component from the mammalian animal.
The term xe2x80x9ccomponentxe2x80x9d as used herein refers to any portion of an animal. A component can be selected from the group consisting of fluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus. Precursor cells may arise from fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses isolated from cloned organisms of the invention.
The term xe2x80x9cgametexe2x80x9d as used herein refers to any cell participating, directly or indirectly, to the reproductive system of an animal. Examples of gametes are spermatocytes, spermatogonia, oocytes, and oogonia. Gametes can be present in fluids, tissues, and organs collected from animals (e.g., sperm is present in semen). For example, methods of collecting semen for the purposes of artificial insemination are well known to a person of ordinary skill in the art. See, e.g., Physiology of Reproduction and Artificial Insemination of Cattle (2nd edition), Salisbury et al., copyright 1961, 1978, WH Freeman and Co., San Francisco. However, the invention relates to the collection of any type of gamete from an animal.
The term xe2x80x9ctissuexe2x80x9d is defined previously. The term xe2x80x9corganxe2x80x9d refers to any organ isolated from an animal or any portion of an organ. Examples of organs and tissues are neuronal tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary and kidney. These examples are not limiting and the invention relates to any organ and any tissue isolated from a cloned animal of the invention.
In a preferred embodiments, the invention relates to (1) fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses can be subject to manipulation; (2) the manipulation can comprise the step of cryopreserving the gametes, embryos, and/or fetal tissues; (3) the manipulation can comprise the step of thawing the cryopreserved items; (4) the manipulation can comprise the step of separating the semen into X-chromosome bearing semen and Y-chromosome bearing semen; (5) the manipulation comprises methods of preparing the semen for artificial insemination; (6) the manipulation comprises the step of purification of a desired polypeptide(s) from the biological fluid or tissue; (7) the manipulation comprises concentration of the biological fluids or tissues; and (8) the manipulation can comprise the step of transferring one or more cloned cells, cloned tissues, cloned organs, and/or portions of cloned organs to a recipient organism (e.g., the recipient organism may be of a different specie than the donor source).
The term xe2x80x9cseparatingxe2x80x9d as used herein in reference to separating semen refers to methods well known to a person skilled in the art for fractionating a semen sample into sex-specific fractions. This type of separation can be accomplished by using flow cytometers that are commercially available. Methods of utilizing flow cytometers from separating sperm by genetic content are well known in the art. In addition, semen can be separated by its sex-associated characteristics by other methods well known to a person of ordinary skill in the art. See, U.S. Pat. Nos. 5,439,362, 5,346,990, and 5,021,244, entitled xe2x80x9cSex-Associated Membrane Proteins and Methods for Increasing the Probability that Offspring Will Be of a Desired Sex,xe2x80x9d Spaulding, issued on Aug. 8, 1995, Sep. 13, 1994, and Jun. 4, 1991 respectively, all of which are incorporated herein by reference in their entireties including all figures, tables, and drawings.
Semen-preparation methods are well known to someone of ordinary skill in the art. Examples of these preparative steps are described in Physiology of Reproduction and Artificial Insemination of Cattle (2nd. edition), Salisbury et al., copyright 1961, 1978, W. H. Freeman and Co., San Francisco.
The term xe2x80x9cpurificationxe2x80x9d as used herein refers to increasing the specific activity of a particular polypeptide or polypeptides in a sample. In preferred embodiments, specific activity is expressed as the ratio between the activity of the target polypeptide and the concentration of total polypeptide in the sample. Activity can be catalytic activity and/or binding activity, for example. In other preferred embodiments, specific activity is expressed as the ratio between the concentration of the target polypeptide and the concentration of total polypeptide. Purification methods include dialysis, centrifugation, and column chromatography techniques, which are well-known procedures to a person of ordinary skill in the art. See, e.g., Young et al., 1997, xe2x80x9cProduction of biopharmaceutical proteins in the milk of transgenic dairy animals,xe2x80x9d BioPharm 10(6): 34-38.
The term xe2x80x9ctransferringxe2x80x9d as used herein refers to shifting a group of cells, tissues, organs, and/or portions of organs to an animal. The cells, tissues, organs, and/or portions of organs can be, for example, (a) developed in vitro and then transferred to an animal. (b) removed from an animal and transferred to another animal of a different specie, (c) removed from an animal and transferred to another animal of the same specie, (d) removed from one portion of an animal (e.g., the leg of an animal) and then transferred to another portion of the same animal (e.g., the brain of the animal), and/or (e) any combination of the foregoing. The term xe2x80x9ctransferringxe2x80x9d refers to adding cells, tissues, and/or organs to an animal and can also relate to removing cells, tissues, and/or organs from an animal and replacing them with cells, tissues, and/or organs from another source.
The term xe2x80x9ctransferringxe2x80x9d as used herein also refers to implanting one or more cells, tissues, organs, and/or portions of organs from the cloned mammalian animal into another organism. For example, neuronal tissue from a cloned mammalian organism can be grafted into an appropriate area in the human nervous system to treat neurological diseases such as Alzheimer""s disease. Alternatively, cloned cells, tissues, and/or organs originating from a porcine organism may be transferred to a human recipient. Surgical methods for accomplishing this preferred aspect of the invention are well known to a person of ordinary skill in the art. Transferring procedures may include the step of removing cells, tissues, or organs from a recipient organism before a transfer step.
In other aspects the invention features (1) a cloned mammalian animal prepared by a process comprising the steps of: (a) preparation of a cloned mammalian embryo by any one of the methods described herein for producing such a cloned mammalian embryo, and (b) manipulation of the cloned mammalian embryo such that it develops into a live born animal; (2) a process comprising the steps of: (a) preparation of a cloned mammalian embryo by any one of the methods described herein for preparing such a cloned mammalian embryo, and (b) manipulation of the cloned mammalian embryo such that it develops into a live born animal; and (3) a cloned mammalian animal, comprising the steps of: (a) preparation of a cloned mammalian embryo by any one of the methods for producing such an embryo described herein, and (b) manipulation of the cloned mammalian embryo such that it develops into a live born animal.
In preferred embodiments, (1) the live born animal is preferably an ungulate animal and more preferably a bovine animal; (2) the manipulation can comprise the step of implanting the embryo into a uterus of an animal; (3) the estrus cycle of the animal can be synchronized to the developmental stage of the embryo; and (4) the manipulation can comprise the step of implanting the embryo into an artificial environment.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.